PROJECT SUMMARY: Hepatocellular carcinoma (HCC), or primary liver cancer, is the 5th most common cancer globally and the 2nd most common cause of cancer death worldwide. HCC is often diagnosed at a late stage at which 85% of patients are not candidates for curative surgical operations; moreover, HCC is notoriously resistant to chemotherapy and other systemic treatments, resulting in a 5-year survival rate of 17.2%. Since cancer is driven by the accumulation of genetic driver mutations conferring selective growth advantages, potential exists for utilization of precise gene-editing techniques to investigate the impact of driver mutations on chemotherapeutic susceptibility for chemoresistant cancers including HCC. This proposal employs the innovative Oncopig Cancer Model (OCM)?a transgenic pig model that recapitulates human cancer through induced expression of heterozygous KRASG12D and TP53R167H driver mutations?to test the hypothesis that compelling phenotypic differences in malignant potential and chemoresistance are induced by accumulation of distinct driver mutations. In this proposal, OCM HCC with distinct driver mutational profiles will be created through CRISPR-Cas9 directed knockout of ARID1A and/or AXIN1?observed in 10-15% of human HCC?on existing KRASG12D and TP53R167H transgene germline mutated OCM cells for investigation of the effects on HCC chemoresistance and malignant potential. AXIN1 mutations result in WNT signaling activation, while ARID1A plays a key role in chromosome remodeling, promoting HCC development, migration, and proliferation. This approach will allow in vitro and in vivo investigation of the contribution of driver mutational profiles on malignant potential and treatment susceptibility in a clinically relevant large animal model with similar anatomy, physiology, metabolism, immunity, and genetics compared to humans. Through assessment of the effects of sequential, spatially controlled driver mutation accumulation on clinically relevant tumor phenotypes, this proposal addresses PQ4: Can we develop tools to directly change the expression or function of multiple chosen genes simultaneously and use these tools to study the range of changes important for human cancer? Our rationale is that the creation and validation of in vivo systems to model HCC will provide a foundation for translational and transformative study of the contribution of driver mutational profiles on clinically relevant phenotypes, ultimately advancing the development of novel, targeted HCC therapeutic approaches. We plan to test our central hypothesis by pursuing the following Specific Aims: (1) To perform simultaneous and stepwise CRISPR-Cas9 directed ARIDA1A and AXIN1 mutation in already established KRASG12D and TP53R167H mutated OCM HCC cell lines, and to define in vitro malignant potential and chemoresistance; (2) To define the in vivo effects of distinct HCC driver mutational profiles on malignancy in a clinically relevant OCM cirrhotic liver microenvironment. This work will set the stage for modeling of additional driver mutation combinations across cancer types and in vivo testing of novel, targeted therapeutic approaches on genotypically defined cancers in a clinically relevant large animal model.